Biological control method for ciliate disease

Abstract

The present disclosure provides a biological control method for ciliate disease, and belongs to the technical field of biological control. The biological control method includes the following step: adding Chaenea vorax into an aquaculture pond to control the ciliate disease; and the Chaenea vorax includes Chaenea vorax PJ13002. According to the present disclosure, the Chaenea vorax PJ13002, a predatory protozoan, is introduced to recover a microbial food chain existing in nature in an aquaculture system and achieve biological control of the ciliate disease. Meanwhile, the Chaenea vorax PJ13002 can be directly ingested by aquaculture animals without causing diseases, so that a more stable aquaculture system can be formed, losses are avoided, costs are reduced, and economic benefits are increased. The method provides a new thought for controlling the ciliate disease and has an excellent popularization and application prospect.

Claims

1. A biological control method for ciliate disease, comprising adding Chaenea vorax to an aquaculture pond to control the ciliate disease, wherein the Chaenea vorax is Chaenea vorax PJ13002 with the deposit number CGMCC NO: 46992.

2. The biological control method according to claim 1, wherein the Chaenea vorax PJ13002 is available from an intertidal zone of Qingdao.

3. The biological control method according to claim 1, wherein the aquaculture pond is maintained at a temperature of 10-20 C., a salinity of 25-35%, and pH 6-9.

4. The biological control method according to claim 1, wherein based on a 30 mu aquaculture pond, the adding Chaenea vorax to an aquaculture pond specifically comprises: if a density of ciliates is 0-5 individuals/mL, adding 4,000-6,000 Chaenea vorax to the aquaculture pond; if the density of ciliates is 6-50 individuals/mL, adding 8,000-12,000 Chaenea vorax to the aquaculture pond; if the density of ciliates is 51-200 individuals/mL, adding 25,000-35,000 Chaenea vorax to the aquaculture pond; if the density of ciliates exceeds 200 individuals/mL, adding 95,000-110,000 Chaenea vorax to the aquaculture pond.

5. The biological control method according to claim 1, wherein the ciliates comprise scuticociliates.

6. The biological control method according to claim 5, wherein the scuticociliates comprise Uronema marinum.

7. The biological control method according to claim 6, wherein the Uronema marinum is Uronema marinum PJ20101A with the deposit number CGMCC NO: 46993.

8. The biological control method according to claim 6, wherein the Uronema marinum PJ20101A is available from an intertidal zone of Qingdao.

9. The biological control method according to claim 1, wherein animals cultivated in the aquaculture pond comprise Scophthalmus maximus, Paralichthys olivaceus, prawns, and flounders.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-B show micrographs of Uronema marinum PJ20101A and Chaenea vorax PJ13002; FIG. 1A shows the Chaenea vorax PJ13002, scale bar=50 m; and FIG. 1B is the Uronema marinum PJ20101A, scale bar=10 m.

(2) FIG. 2 shows results of a feeding experiment of Uronema marinum PJ20101A by Chaenea vorax PJ13002 at 14 C.

(3) FIG. 3 shows results of a feeding experiment of Uronema marinum PJ20101A by Chaenea vorax PJ13002 at 18 C.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(4) The present disclosure provides a biological control method for ciliate diseases: adding Chaenea vorax into an aquaculture pond to control the ciliate diseases. The Chaenea vorax includes Chaenea vorax PJ13002.

(5) The biological control method provided by the present disclosure can transform a simple energy transfer of feed.fwdarw.aquaculture animals in the aquaculture pond into a biological control route of feed.fwdarw.bacteria.fwdarw.pathogenic ciliates.fwdarw.Chaenea vorax.fwdarw.aquaculture animals. This approach creates a green, safe and healthy aquaculture conditions. Moreover, this can increase diversified food sources for aquaculture animals. It effectively reduces diseases and enhances economic benefits in aquaculture operations.

(6) The Chaenea vorax PJ13002 provided by the present disclosure is available from an intertidal zone of Qingdao, where the Chaenea vorax PJ13002 has a deposit number of CGMCC NO: 46992, and was deposited in the China General Microbiological Culture Collection Center (CGMCC) located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, on Jan. 12, 2026.

(7) The Chaenea vorax PJ13002, provided by the present disclosure, can be used for control of scuticociliate diseases in aquaculture, as well as for biological control and killing of ciliates in aquaculture. Meanwhile, the Chaenea vorax can be directly ingested by the aquaculture animals without causing diseases, forming a green aquaculture model based on biological control, reducing diseases and improving economic benefits.

(8) In the present disclosure, the preferred temperature range for the aquaculture pond is 10 to 20 C., with an even more optimal temperature of 18 C. The preferred salinity range is 25 to 35%, with a more optimal salinity of 30%. The preferred pH range is 6 to 9, with a more optimal pH of 8. The predatory ciliate Chaenea vorax PJ13002 provided by the present disclosure has a wide range of adaptability to temperature, salinity and pH, and the growth conditions there of are in line with aquaculture conditions of Scophthalmus maximus, Paralichthys olivaceus, prawns, and flounders, and effective preying can be performed, so as to carry out biological control.

(9) In the present disclosure, preferably based on a 30 mu aquaculture pond, adding Chaenea vorax to an aquaculture pond specifically includes: when the pathogenic ciliate density is between 0 and 5 individuals/mL, the preferred range for introducing predatory ciliates is 4,000 to 6,000 individuals, with a more optimal number of 5,000 individuals. When the pathogenic ciliate density is between 6 and 50 individuals/mL, the preferred range for introducing predatory ciliates is 8,000 to 12,000 individuals, with a more optimal number of 10,000 individuals. When the pathogenic ciliate density is between 51 and 200 individuals/mL, the preferred range for introducing predatory ciliates is 25,000 to 35,000 individuals, with a more optimal number of 30,000 individuals. When the pathogenic ciliate density exceeds 200 individuals/mL, the preferred range for introducing predatory ciliates is 95,000 to 110,000 individuals, with a more optimal number of 100,000 individuals.

(10) In the present disclosure, the ciliates preferably include scuticociliates; the scuticociliates preferably include Uronema marinum; the Uronema marinum is Uronema marinum PJ20101A (the GenBank accession number of the Uronema marinum PJ20101A is OP050455); the Uronema marinum PJ20101A (Uronema marinum) is preferably from an intertidal zone of Qingdao, where the Uronema marinum PJ20101A has a deposit number of CGMCC NO: 46993, and was deposited in the China General Microbiological Culture Collection Center (CGMCC) located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, on Jan. 12, 2026.

(11) In the present disclosure, animals cultivated in the aquaculture pond preferably include Scophthalmus maximus, Paralichthys olivaceus, prawns, and flounders.

(12) The technical solution provided by the present disclosure will be described in detail below with reference to examples, but they should not be construed as limiting the claimed scope of the present disclosure.

Example 1

(13) Isolation, Culture and Identification of Pathogenic Scuticociliates and Predatory Ciliates

(14) The pathogenic Uronema marinum PJ20101A was isolated from the intertidal zone of Qingdao in December 2017. The predatory ciliate Chaenea vorax PJ13002 was isolated in July 2019.

(15) Ulva and seawater were collected from the intertidal zone, temperature, salinity, and pH were measured. A single ciliate was isolated using a mouth pipette under a dissecting microscope. The single cell was passaged to establish a culture system.

(16) Micrographs were taken of Uronema marinum PJ20101A and Chaenea vorax PJ13002. The micrographs are shown in FIGS. 1A-B.

(17) Molecular identification was carried out, and 18S rDNA (primer EukA: 5-AACCTGGTTGATCCTGCCAGT-3 (SEQ ID NO: 1); and primer EukB: 5-TGATCCTTCTGCAGGTTCACCTAC-3 (SEQ ID NO: 2)) fragments were amplified and subjected to Sanger sequencing. Sequencing results were aligned to the NCBI database. Both ciliates were named for the most similarity to Uronema marinum and Chaenea vorax, respectively.

Example 2

(18) Habits of Chaenea vorax PJ13002

(19) Chaenea vorax PJ13002 was tested for the ranges of temperature (14-32 C.), salinity (10-40%), and pH (6-11).

(20) At three temperatures (14, 25, and 32 C.), seven salinities (10%, 15%, 20%, 25%, 30%, 35%, and 40%) and five pH values (6.6, 7.6, 8.6, 9.6, and 10.6), the Chaenea vorax PJ13002 was tested for temperature adaptation range. There were a total of 15 treatments, and there were three parallel samples under each treatment. All conditions were the same except treatment conditions. Three Chaenea vorax PJ13002 were added to each group.

(21) First, 1,000 mL of Escherichia coli suspension (OD=0.3) and approximately 100,000 Uronema marinum PJ20101A were added to a 2 L Erlenmeyer flask successively, and underwent static culture at 25 C. for 4 days. The mixture is incubated at 25 C. for 4 days without agitation. After incubation, thoroughly mix the liquid and prepare 45 Petri dishes with a diameter of 90 mm, each containing 20 ml culture medium.

(22) 1. Temperature

(23) Three temperatures (14 C., 25 C., 32 C.) were set, with three Petri dishes for each temperature. After 20 mL culture medium was added, all Petri dishes were added with three Chaenea vorax PJ13002, and put at the corresponding temperature. The growth state was observed every day, and data statistics were performed according to the average value of the observed quantity of ciliates in the three Petri dishes at each temperature. The specific results are shown in Table 1.

(24) TABLE-US-00001 TABLE 1 Effects of different temperatures on the number of Chaenea vorax PJ13002. Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 14 C. 3 3 11 32 47 74 165 25 C. 3 4 162 887 32 C. 3 3 12 74 154 765 From Table 1, Chaenea vorax PJ13002 could grow at all three temperatures: 14 C., 25 C., and 32 C,; at 14 C., the quantity of ciliates increased slowly, but feeding activities were frequent; at 25 C., the quantity of ciliates increased significantly, and feeding was active; and at 32 C., the quantity of ciliates increased significantly, but feeding was not active.
2. Salinity

(25) Seven salinity levels (10%, 15%, 20%, 25%, 30%, 35%, 40%) were tested, with three Petri dishes set up for each salinity. Each culture dish was filled with 20 ml of sterilized seawater prepared at the respective salinity level (either diluted sterilized seawater or seawater prepared with sea salt); 360 mL of Uronema marinum PJ20101A was centrifuged (at 1,500 g for 5 min) to enrich, and aliquoted into each Petri dish; and three Chaenea vorax PJ13002 were added to each Petri dish and cultured at room temperature, and the growth thereof was observed every day.

(26) The experimental results showed that Chaenea vorax PJ13002 could survive at seven salinities of 10%, 15%, 20%, 25%, 30%, 35%, and 40%, but at low and high salinities of 10%, 15% and 40%, there was no significant increase in the quantity of ciliates, and the feeding activities were reduced; at salinities of 20%, 25%, and 30%, the quantity increased significantly and feeding was active, among which the quantity of ciliates increased most significantly at a salinity of 30%.

(27) 3. pH

(28) Five pH levels (6.6, 7.6, 8.6, 9.6, 10.6) were tested, with three Petri dishes set up for each pH level. After adding 20 mL of culture medium, pH values were adjusted with acid or alkali to make it reach the treatment concentration. Subsequently, three Chaenea vorax PJ13002 were added to each Petri dish and cultured at room temperature, and the growth was observed daily.

(29) The experimental results showed that Chaenea vorax PJ13002 could survive at five pH values, but at pH 6.6 and 10.6, there was no significant increase in the quantity of ciliates; and at pH 7.6, 8.6, and 9.6, the quantity of ciliates increased significantly and feeding was active. Seawater itself is a buffer system that will spontaneously restore the pH value changed by biological growth, providing a stable pH microenvironment for the survival and division of Chaenea vorax PJ13002.

(30) According to the experimental results, Chaenea vorax PJ13002 has a wide range of physiological tolerance to temperature, salinity, and pH value; the growth conditions thereof are in line with aquaculture conditions of Scophthalmus maximus, Paralichthys olivaceus, prawns, and flounders; and effective preying can be performed, so as to carry out biological control.

Example 3

(31) Feeding Experiment of Chaenea vorax PJ13002

(32) The feeding experiment of Chaenea vorax PJ13002 was carried out at two temperatures (14 C., the temperature commonly used in mariculture; and 18 C., the temperature corresponding to the outbreak of pathogenic ciliates), and six groups were divided: three control groups without adding Chaenea vorax PJ13002 and three experimental groups with each adding 50 Chaenea vorax PJ13002 were set up at each temperature.

(33) First, six 100 mL Erlenmeyer flasks were used, and each flask was filled with 40 mL of Escherichia coli suspension (OD=0.3) and approximately 4,000 Uronema marinum PJ20101A were added to each flask successively. After static culture at 14 or 18 C. for two days, 50 Chaenea vorax PJ13002 were added to each Erlenmeyer flask in the experimental groups and thoroughly shaken every 24 h for sampling and counting. The densities of Uronema marinum PJ20101A and Chaenea vorax PJ13002 were measured.

(34) The density measurement method was as follows: using a Gridded Sedgewick Rafter (1 mm.sup.2; 400 L of culture medium+100 L of Bouin's Fixative), 100 L of well-mixed fixative was pipetted and counted (N) under a dissecting microscope. The density in each Erlenmeyer flask was calculated using the following formula: Density=N/801000=12.5N (individuals/mL).

(35) The average values were calculated for the three control groups and three treatments, resulting in the densities of the control and treatment groups. The treatment density refers to the combined density of Uronema marinum PJ20101A and predatory ciliates PJ13002. Please refer to Tables 2-3 and FIGS. 2-3 for specific results.

(36) TABLE-US-00002 TABLE 2 Statistics of the densities of the control and treatment groups fed at 14 C. Density of the Density of control group the treatment Day (individuals/mL) (individuals/mL) 1 8892 8892 2 29859 36869 3 60823 63043 4 58296 52660 5 56515 45609 6 61542 24497 7 55356 52771 8 63057 55220 9 70771 50632 10 75440 28201 11 64273 17139 12 60756 14613 13 61782 8605 14 59134 16357 15 45738 8457 16 49630 6220 17 44917 2558 18 41584 366 19 34557 1045 20 20316 221 21 11503 406

(37) TABLE-US-00003 TABLE 3 Statistics of the densities of the control and treatment groups fed at 18 C. Density of the Density of the control group treatment group Day (individuals/mL) (individuals/mL) 1 8892 8892 2 67267 48181 3 78355 70457 4 91978 83681 5 78922 79596 6 86705 72800 7 71901 31785 8 77436 6643 9 79557 2095 10 75017 297 11 81296 86 12 76555 60 13 76660 49 14 52875 109 15 43172 156

(38) It can be seen from Tables 2 and 3 and FIGS. 2 and 3 that Chaenea vorax PJ13002 can effectively reduce the quantity of Uronema marinum PJ20101A in the culture system and achieve the biological control effect.

Experimental Example 1

(39) Field Test

(40) After an outbreak of scuticociliates (density 150-200 cells/mL; causing the disease of scuticociliatosis) in a 30 mu shrimp pond in Tianjin, 30 mL of the suspension of Chaenea vorax PJ13002 obtained in Example 3 was added to each of the four corners of the shrimp pond, after 7 days, it was observed that the quantity of scuticociliates in the affected pond was significantly reduced (to a density of 1-3 individuals/mL). Every 7 days, 5 mL of the suspension of Chaenea vorax PJ13002 obtained in Example 3 was added to each of the four corners of the shrimp pond, and the scuticociliates were always controlled at a very low density (0-1 individuals/mL). Finally, the shrimp yield of the pond was 850 individuals/mu.

(41) According to past experience, after the outbreak of scuticociliate disease in uncontrolled prawn ponds, all prawns in the entire pond died and the output was not obtained.

(42) It can be known from the field test that the biological control method provided by the present disclosure can reduce the quantity of scuticociliates in the aquaculture pond and achieve the control effect of ciliate disease.

(43) The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.